![Ca v 1-mediated Ca 2+ infl ux induces neuronal progenitor cells (NPCs) to differentiate into neuronal cells. NPCs from Day 4 of differentiation derived from carcinoma (CSC) and mesenchymal (MSC) stem cells were cultured for 2 days in the presence or absence of nifedipine (5 μm) or Bay-K (10 μm), and the percentage of nestin-positive (represented as gray bars) or NEL-positive (represented as dark-gray bars) cells was analyzed by immunofl uorescence, counted, and plotted as a percentage of total cells (A). Gene (B) and protein (C) expression of NEL and nestin from NPC from CSC and MSC at the same conditions were also evaluated. The percentage of cells exhibiting [Ca 2+ ] i transients in response to KCl stimulation was determined (D) and the amplitude of the recorded signals was then evaluated (E).](https://www.researchgate.net/profile/Alexandre-Kihara/publication/23495932/figure/fig1/AS:667194388135949@1536082946644/Ca-v-1-mediated-Ca-2-infl-ux-induces-neuronal-progenitor-cells-NPCs-to-differentiate_Q320.jpg)
Content uploaded by Alexandre Hiroaki Kihara
Author content
All content in this area was uploaded by Alexandre Hiroaki Kihara on May 31, 2015
Content may be subject to copyright.
1
STEM CELLS AND DEVELOPMENT
Volume XX, Number XX, 2009
© Mary Ann Liebert, Inc.
DOI: 10.1089/scd.2008.0289
Intracellular Ca2 Regulation During Neuronal Differentiation of
Murine Embryonal Carcinoma and Mesenchymal Stem Cells
Rodrigo R. Resende,1,2 José L. da Costa,3 Alexandre H. Kihara,4,5 Avishek Adhikari,6 and Eudes Lorençon1
Changes in intracellular Ca2 concentration ([Ca2
]i) play a central role in neuronal differentiation. However,
Ca2 signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem
cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two
cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells
(MSC). We studied Ca2 release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and
IP3-sensitive (IP3R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca2
]i response that increased
throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L- but not
with N-, P-, or Q-type Cav1 Ca2 channels, both in embryonal CSC and adult MSC. We also found that agonists
of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these
channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca2
release following voltage-dependent Ca2 entry via L-type Ca2 channels. This study shows that both in embry-
onal CSC and adult MSC [Ca2
]i is controlled by a common pathway, indicating that coupling of L-type Ca2
channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.
Introduction
C physiological roles [1]
including intercellular signaling [2], proliferation [3],
apoptosis [3], control of gene expression [4], and release of
bioactive molecules such as neurotransmitters [5,6] due to
calcium in ux from voltage-gated Ca2 channels (VGCCs)
[7]. Interestingly, Ca2 in ux through VGCCs, such as long-
lasting Ca2 channels (L-type, also known as Ca
v1), also
affects the expression of genes involved in cell proliferation,
programmed cell death, and neuronal differentiation [8–11].
Considering that calcium is involved in this multitude of
processes, it is not surprising that intracellular Ca2 con-
centration ([Ca2
]i) is controlled by multiple mechanisms.
Calcium in ux may occur through membrane channels as
well as through intracellular stores such as the endoplas-
mic reticulum (ER). Release of Ca2 from the ER is simul-
taneously controlled by ryanodine receptors (RyRs) [12]
and inositol-1,3,4-triphosphate receptors (IP3Rs) [13], and
counterbalanced by the activity of Ca2 pumps such as
sarco/endoplasmic reticulum Ca2-ATPase (SERCA) [14].
Calcium in uxes through these intracellular stores may
participate in neuronal differentiation, a process in which
the role and pathways of Ca2 signaling have been exten-
sively i nvestigated and modeled [15–19]. However, it is not
clear whether particular Ca2 pathways are cell-line-speci c
or if there are common processes in the neuronal differ-
entiation of diverse cells lines. We thus characterized Ca2
signaling in stem cell lines as distinct as embryonal carci-
noma stem cells (CSC) and adult murine bone-marrow mes-
enchymal stem cells (MSC), to identify conserved events in
the control of cell proliferation and differentiation. We have
previously shown that P19 embryonic CSC express func-
tional VGCCs, which produce Ca2-induced Ca2 release
(CICR) through a combination of L-type Ca2 channels and
RyRs and IP3Rs activity [19,20]. We now report that the RyR
agonist caffeine produces a transient increase in Ca2 that
1
Department of Physics, Institute of Exacts Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil.
2
Instituto de Ensino e Pesquisa Santa Casa de BH (ISCM-BH), Belo Horizante, Brazil.
3
Instrumental Analysis Laboratory, Criminalistic Institute of São Paulo, São Paulo, Brazil; Institute of Chemistry, University of São
Paulo, São Paulo, Brazil.
4
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
5
Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, Santo André, Brasil.
6
Department of Biological Sciences, Columbia University, New York.
SCD-2008_0289.indd 1 3/10/2009 4:55:41 PM
RESENDE ET AL.2
BMCs were stained with primary rat–anti-mouse antibod-
ies, namely anti-CD4, CD8, CD11b, CD45R/B220, Gr-1, and
Ter119 (all purchased from PharMingen, San Jose, CA). After
centrifugation of the labeled cells, Dynabeads M-450 Sheep
anti-Rat IgG (Dynal Biotech, Oslo, Norway) were added at a
concentration of 10 beads per cell. The mixture was placed on
a Dynal Magnetic Particle Concentrator (Dynal Biotech) for 1
min and the supernatant containing the line-depleted BMCs
was collected. Immunodepleted BMCs were resuspended
in 10 mL of complete medium and plated on a 35 10 mm
tissue culture dish (NUNC, Naperville, IL). Cells were cul-
tured in a humidi ed incubator containing 5% CO2 and 5%
O2 at 37°C. After 24 h, nonadherent cells were removed by
washing with PBS and a fresh complete medium was added.
Afterwards, the medium was changed twice a week for 3–4
weeks, when cells rst reached 70–80% con uency. Then,
adherent cells were detached with trypsin/EDTA for 3 min
at 37°C. Only easily detached cells were plated on a 35
10 mm standard tissue culture dish ( rst passage). After the
rst passage, cells were cultured under similar conditions,
except that the split ratio was set to 1:2. After additional 5–7
passages, cells were detached at a lower cell density (40–50%
con uence). The same conditions were used for subsequent
cultures after small rou nd and oval-shaped cells had become
predominant by the sixth to eighth passage.
A total of 1 105 cells was suspended in complete
medium in each well of a six-well ultra low attachment plate
(NUNC, Naperville, IL). Cells were cultured without hav-
ing the medium changed, and were rotated gently every 6
h. The spheres gradually increased in size and, 7 days after
suspension, were picked and dissociated mechanically by
gentle pipetting. Dissociated cells were cultured in complete
medium on standard tissue culture dishes and the medium
was changed every 2 days. After reaching 40–50% con u-
ency, cells were detached with trypsin/EDTA and replated
at a 1:4 dilution. Sphere-derived cells were harvested for the
experiments described below when a single well of the orig-
inal culture contained >1 107 cells.
In order to induce neuronal differentiation, sphere-
derived cells were treated as previously described [24] with
some modi cations. Brie y, cells were plated at a cell density
of 1 104 cells/cm2 on gelatin-coated dishes in neurobasal
medium (Gibco BRL, Grand Island, New York) with B27
Supplement (50; Invitrogen Corp., Carlsbad, CA), 20 ng/
mL EGF, and 10 ng/mL bFGF for 7 days, followed by culture
in neurobasal medium with 0.5 M retinoic acid and 20 ng/
mL -NGF for 5 days.
Immuno uorescence studies
For immuno uorescence, cells were xed in 1%
paraformaldehyde (PFA), permeabilized with 0.2% Triton
X-100 in PBS at 4°C for 30 min, blocked with 0.5% casein
and 5% normal goat serum in PBS at 4°C for 30 min, and
sequentially incubated with anti-nestin (1:200; Chemicon
International Inc., Temecula, CA) or NEL (1:100; Santa
Cruz Biotechnology Inc., Delaware, CA, USA) antibodies
at 4°C overnight, followed by incubation with secondary
antibodies coupled with cytochrome 3 (Cy3, 1:200; Jackson
Immunoresearch, West Grove, PA) at room temperature (RT)
for 1 h. Between each step, cells were washed three times
with 0.1% Twee n in PBS. Fluore scence was dete cte d and pho -
tographed with an AxioCam photomicroscope (Carl Zeiss
becomes larger throughout neuronal differentiation both in
CSCs and MSCs. Moreover, these Ca2 transients are blocked
by L-type Ca2-channel blockers, suggesting that RyRs and
L-type Ca2 channels are coupled. Our data also show that
agonists of both of these channels increase neurogenesis
and differentiation into neurons in CSCs and MSCs, while
antagonists of these channels block these processes. Thus,
this report suggests that coupling of L-type Ca2 channels
and RyRs is a mechanism that may be conserved in neuro-
nal differentiation in diverse stem cell lines, and the IP3Rs
have a major role in induction of neuronal differentiation
beyond the RyRs.
Materials and Methods
Reagents
Unless indicated otherwise, all reagents were purchased
from Sigma (Sigma, St. Louis, MO). Appropriate secondary
biotin conjugate antibodies were used at 1/200 dilutions
(Santa Cruz Biotechnology, Heidelberg, Germany). Primers
for real-time PCR were synthesized by Integrated DNA
Technologies (Coralville, IA).
Culture and neuronal differentiation of CSC cells
P19 EC cells were grown in DMEM (Invitrogen Corp.,
Carlsbad, CA) supplemented with 10% of nal volume with
fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 100
units/mL penicillin, 100 g/mL streptomycin, 2 mM sodium
pyruvate, and 2 mM -glutamine in a humidi ed incubator
at 5% CO2 and 37°C. One micromolar of all trans-retinoic acid
(RA) was used for neuronal differentiation induction. P19
cells were cultured in suspension to induce the formation
of embryonic bodies (EB) in bacterial culture dishes coated
with 0.2% of nal volume with agarose for 48 h at a den-
sity of 5 105 cells/mL in de ned serum-free medium as
described previously [18,19,21,22]. Glial cell growth was sup-
pressed by treatment with 50 g/mL cytosine arabinoside
on day 6 of differentiation.
Isolation and culture of sphere-derived cells from
murine bone marrow
All experiments in this study were performed in accor-
dance with the Animal Protection Guidelines of University
of São Paulo. Bone-marrow cells (BMCs) were obtained
from ve mice for each experiment as previously described
[23]. Brie y, bone marrow was collected from 2-month-old
C57Bl/6 mice by ushing femurs and tibias with complete
medium constituted of Dulbecco’s modi ed Eagle’s medium
(DMEM), 10% fetal calf serum (FCS; Sigma, St. Louis, MO),
2 mM glutamine and 50 U/mL penicillin, 50 mg/mL strep-
tomycin supplemented with heparin at a nal concentration
of 5 U/mL. Cells were then washed twice in a heparin-free
medium and plated in a Petri dish at a density of 2 106
cells/cm2. After 2 days, nonadherent cells were removed
by two to three washes with phosphate-buffered saline
(PBS) and adherent cells were further cultured in complete
medium for four supplementary days. To remove differen-
tiated hematopoietic cells, these cells were depleted from
whole BMCs by means of immunomagnetic separation with
Dynabeads according to the manufacturer’s protocol. Brie y,
SCD-2008_0289.indd 2 3/10/2009 4:55:41 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 3
Western-blot assays
NPC from day 6 derived from CSC and MSC were homog-
en ized in ice -cold lys is buffer cont aini ng pro tease inhibito rs,
10 mM Tris-HCl (pH 7.5), 8 M urea, 20 mM EDTA, 150 mM
NaCl, 1% Triton X-100. Forty micrograms of membrane pro-
teins that were fractionated by differential centrifugation,
separated by SDS-PAGE, and transferred to nitrocellulose
membranes were use d. The procedure of incubation with pri-
mary and secondary antibodies was essentially the same as
described previously [18,21]. The following polyclonal rabbit
antisera were used: anti-nestin (1:200), anti-neuronal enolase
(NEL, 1:100), anti-SSEA-1 (1:100; Santa Cruz Biotechnology,
Heidelberg, Germany), and anti--actin (1:400). -actin pro-
tein expression was used as internal standard for relative
quanti cation of nestin and NEL expression levels.
RNA isolation, reverse transcription, real-time PCR,
and conventional PCR
Total RNA was isolated using TRIzol (Invitrogen Corp.,
Carlsbad, CA) from undifferentiated cells and from days
2 to 8, following the induction of neuronal differentiation
in the presence of RA for CSC and in the presence of RA
and -NGF for MSC. Additional RNA was extracted on the
sixth day of neuronal differentiation that had been cultured
for 48 h in the presence or absence of 5 M nifedipine or
10 M Bay-K. Integrity of the isolated RNA was veri ed by
separation of an aliquot of the extracted RNA on a 2% ethid-
ium bromide-stained agarose gel. DNA was removed from
RNA samples by incubation with DNase I (Ambion Inc.,
Aust in, TX).
Three micrograms of total RNA were reverse tran-
scribed to cDNA with 200U of RevertAid H Minus M-MuLV-
reverse transcriptase (Fermentas Inc., Hanover, MD). DNA
templates were ampli ed by real-time PCR on the 7000
Sequence Detection System (ABI Prism; Applied Biosystems,
Foster City, CA) using the Sybr green method [19,22] or were
ampli ed by PCR and analyzed as described previously
[18,19,22]. Variations in cDNA concentrations were normal-
ized with -actin as an internal control, which is a consti-
tutively expressed gene. Experiments were performed in
triplicate for each data point. Primer sequences for reverse
transcription and quantitative PCR ampli cation (qRT-PCR)
of -actin, IP3Rs and RyRs isoforms mRNA and for PCR
ampli cation (RT-PCR) of -actin, nestin, and NEL cDNA
used in this study are listed in Table 1. Negative controls
were conducted on water and on total RNA nonreverse tran-
scribed. To evaluate the speci city of the RT-PCR products,
they were gel-puri ed, cloned into PGEM T-Easy vectors
(Promega, Madison, WI), sequenced, and con rmed to be
identical to mouse NEL and nestin cDNA.
Proliferation assays
5-B rom o-2- de oxy ur idine (Br dU; A mers ha m Li fe Sc ie nce s,
Arlington, IL, EUA), a pyrimidine analog, was used to deter-
mine the synthesis rate of DNA and to perform an incor-
poration assay. Twenty-four hours prior to proliferation
stimulation, NPC were kept in serum-free medium. They
were then maintained for 8 h in culture medium in the
presence or absence of 1 M ryanodine (Rya ExC, acts as
an activator of ryanodine-sensitive calcium stores) or 10 M
Vision GmbH, Hallbergmoos, Germany). The immuno-
uorescent staining of a single sphere was performed as
previously described [25].
Confocal [Ca2]i imaging
[Ca2]i was measured in single cell as previously
described [19,21,22]. The undifferentiated and differentiat-
ing cell cultures were incubated with 2.5 M Ca2-sensitive
dye Fluo3-AM (dissolved in DMSO) plus 0.02% Pluronic
F-127 (dissolved in water; Molecular Probes Inc., Eugene,
OR) in DMEM medium supplemented with FBS for 30 min
at 37°C. The osmolarity of all the solutions ranged between
298 and 303 mosmol/L. Measurements of [Ca2]i were per-
formed at 37°C in extracellular medium (EM) containing
(in mM): 140 NaCl, 3 KCl, 1 MgCl2, 2.5 CaCl2, 10 HEPES,
pH 7.4, 10 glucose with the Zeiss Microscope Photometer
System (FFP; Carl Zeiss Optronics GmbH, Oberkochen,
Germany), based on an inverted microscope (Axiovert 100;
Zeiss). Fluo3 uorescence was excited with a 488 nm line
of an argon ion laser, and the emitted light was detected
using a bandpass lter at 515–530 nm. Fluorescence values
were corrected for background and dark current. Calcium
imaging was performed with a LSM 510 confocal micro-
scope (Carl Zeiss Jena GmbH, Jena, Germany), using image
sizes of 256 256 pixels and acquisition rates of one frame
per second. The calibration of the uorescence signal, in
terms of free Ca2 concentration, was based on the proce-
dure described by Grynkiewicz and collaborators [26]. At
the end of each experiment, 5 M ionophore (4-Br-A23187)
followed by 10 mM EGTA were used to determine Fmax and
Fmin uorescence values, respectively. The average baseline
uorescence was normalized to Fmin as p re vi o us ly de s cr i be d
[19,21]. To evaluate the contribution of VGCC activation to
the studied Ca2 signals, the depolarizing stimuli were
delivered in the presence of the nonselective VGCC blocker
Cd2 (250 µM). Before testing the effects of Cd2, the stabil-
ity of [Ca2]i responses to depolarizing stimuli repeated at
5-min intervals was evaluated. The amplitude of the F/F
ratios in response to the rst four stimulations remained
fairly stable; those of the third and fourth transients were,
respectively, 4.9 2.3 % (n 45) and 8.5 2.1 % (n 37)
smaller than that of the rst.
The role of Cav1-mediated Ca2 in ux in promot-
ing neuronal differentiation of neuronal progenitor cells
(NPC) derived from CSC and MSC was also investigated.
Depolarization-induced Ca2 transients were studied in
cells cultured in de ned medium with the addition of either
5 µ nifedipine or 10 µ Bay-K, a speci c L-type channel
blocker and an activator, respectively. Control cells were
cultured in medium containing the same concentration of
ethanol present in dihidropiridine (DHP)-treated cultures.
Before being used in Ca2-imaging experiments, cells were
repeatedly washed and subsequently subjected to a 10-min
drug-washout perfusion with DHP-free solution to prevent
any acute effects of DHP on the signals being studied.
For calcium imaging in Ca2-free extracellular medium,
cells were treated for 1 min with Ca2-free medium contain-
ing 1 mM EGTA. Control measurements in the presence
of extracellular Ca2-containing medium were performed
before and after experiments in Ca2-free medium. All
antagonists were preincubated with CSCs or MSCs for, at
least, 20 min before experiments were performed.
SCD-2008_0289.indd 3 3/10/2009 4:55:42 PM
RESENDE ET AL.4
neutralization with 0.1 M sodium tetraborate, and incuba-
tion with Alexa Fluor 647-conjugated anti-BrdU (Molecular
Probes Inc., Eugene, OR) monoclonal antibody for 1 h at
room temperature. After washing with PBS, BrdU/NEL, or
BrdU/nestin, stained cells were examined by uorescence
microscopy [18,21].
Statistical analysis
Data are represented as means SEM of ve or more
independent experiments (each with two replicates). The
peak amplitude of [Ca2
]i increase/response is represented in
the bar diagrams with mean SEM. Statistical signi cance
was determined by Student’s t-test or one-way ANOVA plus
a post-hoc Tukey’s test. Values of p < 0.05 wer e considered as
statistically signi cant.
Results
Resting [Ca2]i levels
Basal [Ca2
]i levels were 31 3.9 nM in embryonic CSCs
(n 101) and 72 8.4 nM in the eighth day of neuronal-
differentiated CSCs (n 98). For undifferentiated MSCs,
basal [Ca2
]i levels were 47 5.7 nM (n 86) and 102 12.6
nM after neuronal phenotype acquisition (n 74). Unde r
resting conditions, the basal (resting) [Ca2
]i levels in these
differentiating cells varied, depending on the day of culture,
from 51 5.0 nM (CSC, n 48) and 62 6.7 nM (MSCs,
n 57) on the fourth day after RA or RA and -NGF addi-
tion, respectively, to 74 9.8 nM (n 64) and 87 10.1 nM
(n 72) on the sixth day. Although small variations of the
ryanodine (Rya InhC, an inhibitor of ryanodine-sensitive
calcium stores), which were co-applied with or without ago-
nists or inhibitors of receptor-induced calcium signal trans-
duction. BrdU (15 µM) was then added to the cell culture and
kept for an additional hour. Cell proliferation was detected
by using an anti-BrdU monoclonal antibody (Roche Applied
Science, Indianapolis, IN), biotinylated goat a nti-mouse IgG,
and avidin–biotin complex (Jackson Immuno Research,
West Grove, PA; 1:200). Aminoethylcarbazole (AEC) was
used as a chromogen for visualization of immunostained
cells. Further details are provided elsewhere [18,21].
The effects of Rya ExC, Rya InhC, 10 M Bay-K, an ago-
nist of L-type Ca2 channels, 5 M nifedipine, a blocker of
VGCCs, 10 M cyclopiazonic acid (CPA), a sarcoplasmatic/
endoplasmatic reticulum Ca2-ATPase (SERCA) inhibitor, 10
M xestopongin C (XeC), a membrane-permeable inhibitor
of IP3 and of SERCA pumps [27,28], adenophostin-A as ago-
nist for IP3Rs [29,30], and 20mM KCl as a depolarizing agent
were evaluated for proliferation and neuronal differentia-
tion induction on NPC.
In immuno uorescence double-labeling experiments,
NPC were xed in acid alcohol and processed for NEL or
nestin staining, followed by BrdU staining. Cell prepara-
tions were then incubated with rabbit anti-NEL or anti-nes-
tin antibodies (1:100; Santa Cruz Biotechnology, Delaware,
CA), and immuno uorescence was detected in the presence
of anti-rabbit IgG-Cy3 (Abcam, Cambridge, MA) or IgG-
Alexa-Fluor 488 (Molecular Probes Inc., Eugene, OR) sec-
ondary antibodies. Incubations were performed for 1 h at
room temperature. NEL or nestin immunostaining was fol-
lowed by incubation of the cell preparation with 1 N HCl,
T 1. P A IP3R RyR R S
R-T C PCR
Gene
Access
number Primer Sequence (5–3)Length (bp)
qRT-PCR
IP3R1 NM_010585 FWD 5-CTGCTGGCCATCGCACTT-366
REV 5-CAGCCGGCAGAAAAACGA-3
IP3R2 NM_019923 FWD 5-AGCACATTACGGCGAATCCT-377
NM_0105868 REV 5-CCTGACAGAGGTCCGTTCACA-3
IP3R3 NM_080553 FWD 5-CGGAGCGCTTCTTCAAGGT-375
REV 5-TGACAGCGACCGTGGACTT-3
RYR1 NM_009109 FWD 5-AGACGCTACCACCGAGAAGAAC-383
REV 5-TGGAAGGTGGTTGGGTCATC-3
RYR2 NM_023868 FWD 5-CCGCATCGACAAGGACAAA-376
REV 5-TGAGGGCTTTTCCTGAGCAT-3
RYR3 NM_177652 FWD 5-CGCCTGAGCATGCCTGTT-393
REV 5-TTCTTGCATCTGTTTCCTTTTTTG-3
-Act in NM_007393 FWD 5-GACGGCCAGGTCATCACTATTG-366
REV 5-AGGAAGGCTGGAAAAGAGCC-3
RT-PCR
Nest i n NM _016701 F WD 5’- GAGAGTCGCTTAGAGGTGCA- 3’ 241
RE V 5’- CC ACTTCCAGACTCCTTTAC-3’
NEL NM_013509 FWD 5’-TTGGAGCTGGTGAAGGAAGCC-3’ 486
REV 5’-CGTTCATTGCTGATCTTGTC-3’
-Act in NM_007393 FWD
REV
5-AGGAAGAGGATGCGGCAGTGG-3
5-CGAGGCCCAGAGCAAGAGAG-3
535
FWD, forward primer; REV, reverse primer.
SCD-2008_0289.indd 4 3/10/2009 4:55:42 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 5
the majority of the cells express neuronal progenitor mark-
ers), sixth day (start of neuronal maturation), and eighth day
(when mature neurons are the main population) after induc-
t ion of n eu ro na l di ff er ent ia ti on [ 22, 32]. As we h ave pre vi ousl y
shown, caffeine did not induce a [Ca2
]i response in embry-
onic CSCs [19]. Caffeine-induced [Ca2
]i responses in differ-
entiating cells were 381 7.0 nM on the fourth day and 712
17.9 nM on the sixth day after RA induction and increased
to 945 34.8 nM after the eighth day. MSC [Ca2
]i responses
induced by caffeine were, respectively, 304 13.5 nM, 566
57.2 nM, and 818 12.0 nM on days 4, 6, a nd 8 after induction
of differentiation. Caffeine-induced [Ca2
]i responses dur-
ing differentiation presented an increase in the [Ca2
]i from
embryonic to neuronal mature cells from the eighth day in
culture, when the maximum response was observed (Fig. 2).
Intracellular calcium stores activated by CPA
IP3Rs activation induces Ca2 release from intracellular
calcium stores in embryonic and neuronal cells [28,33–36].
In order to check the involvement of Ca2 release from IP3-
sensitive stores, we have tested the [Ca2
]i response induced
by 10 µM of CPA applied during 30 s at different days in
vitro. CPA (10 M) was applied for 30 s on cell cultures
on days 0, 4, 6, and 8 of differentiation derived from both
CSCs and MSCs. First, the CPA-induced [Ca2
]i response
was nearly identical when applied twice after a long wash
between applications to CSC or MSC. However, these [Ca2
]i
responses had constant [Ca2
]i values throughout the dif-
ferentiation process, differently from the caffeine-induced
resting [Ca2
]i levels were observed, no clear correlation
could be found with the number of days cells were in cul-
ture. Data pooled from days 0 to 8 were scattered around a
mean value of 57 6.8 nM (CSC, n 52) and 74 8.8 nM
(MSC, n 61).
Gene expression of RyR and IP3R isoforms during
neuronal differentiation
We next studied gene expression of IP
3R and RyR iso-
forms during neuronal differentiation of CSCs and MSCs.
At 8 days in vitro, among the three RyR isoforms, only RyR3
and RyR2 were detected in signi cant levels, in CSCs and
MSCs, respectively. All three IP3R isoforms were detected,
although IP3R type 1 and 2 mRNA expressions were very
weak in CSCs and MSCs, respectively (Fig. 1). Real-time PCR
revealed that gene expression of IP3R 1, 2, and 3 subtypes
was already present in embryonic CSCs and adult MSCs,
remaining at a lower level during initial and intermediate
differentiation states and increasing signi cantly when dif-
ferentiating cells became post-mitotic and underwent nal
maturation on days 6–8 (Fig. 1).
[Ca2]i responses induced by caffeine increase
during neuronal differentiation
Caffeine (10 mM), which in millimolar concentrations
acts as an inhibitor of intracellular receptors for IP3, as well
as an activator of RyRs [31], was applied for 10 s on undiffer-
entiated and differentiated cells from the fourth day (when
Differentiation (days)
D0
MSC
4
2
0
2
4
6
8
*
*
*
* *
CSC
10
12
D2 D4 D6 D8 D10
Norm. mRNA exp. (2n)
RyR1
Differentiation (days)
D0
4
2
0
2
4
6
8 MSC
CSC
10
12
D2 D4 D6 D8 D10
Norm. mRNA exp. (2n)
IP3R1
*
*
* *
*
MSC
CSC
4
2
0
2
4
6
8
10
12
Differentiation (days)
D0 D2 D4 D6 D8 D10
Norm. mRNA exp. (2n)
RyR2
* * * *
*
*
MSC
CSC
4
2
0
2
4
6
8
10
12
Differentiation (days)
D0 D2 D4 D6 D8 D10
Norm. mRNA exp. (2n)
RyR3
IP3R2
Differentiation (days)
D0
4
2
0
2
4
6
8 MSC
CSC
10
12
D2 D4 D6 D8 D10
Norm. mRNA exp. (2n)
IP3R3
Differentiation (days)
D0
4
2
0
2
4
6
8 MSC
CSC
10
12
D2 D4 D6 D8 D10
Norm. mRNA exp. (2n)
FIG. 1. Quanti cation of the different isoforms of ryanodine (RyR) and inositol-1,4,5-triphosphate (IP3R) re ceptor mRNA
transcription during neuronal differentiation of carcinoma (CSC) and mesenchymal (MSC) stem cells by real-time PCR.
Gene expression levels of receptor subtypes were normalized to the internal control -actin. The graphs show RyR and IP3
mRNA transcription levels in differentiating cells compared to those in undifferentiated cells which were normalized to
zero. Cells on Day 8 of differentiation were pretreated on Day 6 with cytosine arabinoside, in order to avoid contamination
of the cell cultures with glial cells. The data shown are mean values SEM of at least ve independent experiments in trip-
licate. In some cases, the error bar is included within the symbol. *p < 0.05 compared with control data.
SCD-2008_0289.indd 5 3/10/2009 4:55:42 PM
RESENDE ET AL.6
a very small increase in [Ca2
]i to 105 15.7 nM (CSC-DN)
and 123 10.2 nM (MSC-DN). After preincubation with RyA
InhC, caffeine induced a very small [Ca2
]i elevation in neu-
rons derived from both, CSCs ([Ca2
]i 47 6.1 nM) and
MSCs ([Ca2
]i 164 17.8 nM). No signi cant inhibition of
[Ca2
]i increases was obtained with the IP3R inhibitor XeC
(10 M) on both, CSC and MSC. These results indicated that
Rya InhC inhibited caffeine-induced [Ca2
]i responses.
In order to evaluate the speci city of CPA activity on
intracellular calcium stores, XeC (10 M) was used to inhibit
IP3Rs and SERCA pumps (data not shown). CPA-induced
[Ca2
]i responses were of 242 38.6 nM and 254 27.2 nM
on CSC-DN and MSC-DN, respectively, and these responses
were practically absent when the culture cells were preincu-
bated with XeC for 5 min ([Ca2
]i in nM: 17 2.2 and 35
10.7, respectively). The inhibition of the IP3Rs and the SERCA
pumps responses caused by the presence of XeC was revers-
ible as receptor responses recovered following washing out
of the inhibitor ( [Ca2
]i in nM: 234 35 for CSC-DN and
242 29 for MSC-DN).
[Ca2]i responses induced by Cav1 increase during
neuronal differentiation
To further characterize L-type Cav1 channel and RyR
[Ca2]i responses, 10 µM Bay-K, an agonist of L-type chan-
nel s, or 1 µM r ya nodi ne (Rya ExC) were appl ied. Each drug
calcium transients, which increased progressively across
days (Fig. 2).
Spontaneous [Ca2
]i oscillations were observed under
resting conditions or after a brief application of caffeine
throughout the differentiation. Neuronal-differentiated
(eighth day) cells showed a mean [Ca2
]i peak amplitude of
about 2.6 0.03 for CSC- and 3.6 0.05 for MSC-derived
neurons (DN). These responses were diminished when
cells were incubated with 5 M nifedipine (N- or P/Q-type
Ca2-channel blockers had no effect—data not shown) or 1
M thapsigargin (inhibitor of intracellular calcium stores),
recovering to normal levels after washout of these drugs
(data not shown).
Caffeine- and CPA-speci city action
Caffeine-induced [Ca2
]i responses were initially tested in
the presence of 10 M ryanodine to verify the speci city of
caffeine action on the [Ca2
]i increases. Ryanodine at a con-
centration of 10 M (ryanodine inhibitory, or Rya InhC) acts
as an inhibitor of RyRs, while 1 M ryanodine (ryanodine
excitatory, or Rya ExC) excites RyRs. Neuronal-differentiated
cells from the eighth day were used for this test. In control
conditions, caffeine-induced [Ca2
]i responses were (in nM):
100 8 50.4 and 957 56.0 for CSC- and MSC-DN, respec-
tively (data not shown). To avoid any additional [Ca2
]i ele-
vations ryanodine alone was applied for 2 min, inducing
Day 0
CSC
0
200
400
600
CPA
CPA
CSC
Day 0
Caff
Day 4
Caff
Day 6
Caff
Day 8
Caff
CPA CPA CPA
50 s
50 s
1F/F0
[Ca2]i (nM)
**
**
**
**
**
**
** *
**
*
Caffeine
800
1000
MSC CSC MSC CSC MSC CSC MSC
Day 4 Day 6 Day 8
FIG. 2. Patterns of caffeine
and cyclopiazonic CPA-
induced [Ca2
]i increases dur-
ing neuronal differentiation
of carcinoma (CSC) and mes-
enchymal (MSC) stem cells,
respectively. Upper panel:
Representative traces of
[Ca2
]i responses of CSC cells
after application of caffeine
(Caff, 10 mM) and then with
the SERCA inhibitor cyclo-
piazonic acid (CPA, 10 M).
Lower panels indicate values
of ∆[Ca2
]i increases obtained
in the presence of caffeine
represented with gray bars
or CPA represented with
black bars. Similar observa-
tions were obtained in ve
different experiments for
each cell line. The total num-
ber of cells analyzed in each
experiment was 51–63. Data
are presented as mean values
SEM; *p < 0.05, **p < 0.001
compared with control data.
SCD-2008_0289.indd 6 3/10/2009 4:55:42 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 7
channel), -CgTx (for N-type), and -MVIIC (for P/Q-type
Cav2 channels), were used to evaluate the participation of
these channels in caffeine-evoked [Ca2
]i increases (data
not shown). These VGCC blockers were preincubated with
the cells for 15 min at room temperature in the dark prior
to application of caffeine. The rst application of caffeine
evoked an increase of 1016 55.6 nM and 892 45.0 nM
for CSC-DN and MSC-DN, respectively. Caffeine-induced
[Ca2
]i responses were strongly reduced in the presence of
5 M nifedipine in both, CSC-DNs and MSC-DNs ([Ca2
]
i in nM: 246 21.4 and 343 15, respectively). The N-type
channel blocker, -CgTx-GVIA (800 nM), and the P-type
channel blocker, -MVIIC (100 nM), presented no effects on
caffeine-induced [Ca2
]i responses in CSC-DNs (in the pres-
ence of -CgTx-GVIA, [Ca2
]i 1030 46.6 nM; and in the
presence of -MVIIC, [Ca2
]i 987 49.7 nM, relative to
control conditions [Ca2
]i 951 62.7 nM). However, for
MSC-DNs the N-type channel blocker promoted a signi -
cant reduction on [Ca2
]i elevations by caffeine ([Ca2
]i
674 111.7 nM, relative to control conditions [Ca2
]i 862
36.6 nM).
was applied for 30 s on CSC and MSC cells cultures dur-
ing neuronal differentiation. L-type Cav1 channel-induced
[Ca2]i responses increased during the differentiation
process for both, CSCs and MSCs. The [Ca2]i in nM for
embryonic CSCs was 148 12.4, and for differentiating
cells it was 303 15.9, 694 36.2, and 970 36.8 at 4, 6, and
8 days after induction with RA. The response for undiffer-
entiated MSCs was 226 14.7, while it was 419 11.1, 693
18.2, and 885 39.1 at days 4, 6, and 8 following induction
with RA and -NGF (Fig. 3A). As demonstrated for Bay-K
application and con rming real-time PCR and caffeine-
induced [Ca2]i transients data, ryanodine-induced [Ca2]i
responses increased during the differentiation of CSC and
MSC ([Ca 2]i in nM for: embryonic CSC, 17.9 4.9; fourth
day, 290 7.0; sixth day, 723 40.3; and 857 34.8 at eig hth
day. And for MSC: undifferentiated MSC, 15 1.8; fourt h
day, 290 6.9; sixth day, 771 40.6; and 939 13.0 at eighth
day; Fig. 3A).
To elucidate if RyR or L-type Cav1 channel activation con-
trols [Ca2
]i transients of each other, VGCCs were activated
with 20 mM KCl and these responses were signi cantly
inhibited by ryanodine InhC (Fig. 3B). Furthermore, [Ca2
]i
transients mediated by 10 µM Bay-K were also signi cantly
inhibited by 10 µM ryanodine (Fig. 3B). These results indi-
cated that L-type Cav1 channel-induced [Ca2
]i responses
were mainly controlled by RyRs in neuronal-differentiated
cells derived from CSC and MSC, with a minor role played
by N-type channel for the latter.
L-type Cav1 channels are controlled by
caffeine-induced [Ca2]i responses
The role of the various VGCCs expressed by neuronal-
differentiated CSCs and MSCs cells on caffeine-induced
[Ca2
]i responses was studied in order to evaluate the con-
tribution of extracellular Ca2 to neuronal differentiation.
Blockers of VGCCs, such as nifedipine (for L-type Ca
v1
ACSC Day 0
Rya Exc
Bay K
0
CSC
Day 0
KCl
1250 CSC
** ** ** **
** **
MSC
938
625
313
KCl
KCl
Rya Inhc
Rya Inhc
Bay K
Bay K
Nifedipine
Bay K
Bay K
0
KCl
10 M
Ryanodine
10 M
Ryanodine
5 M
Nifedipine
Bay K Bay K Bay K Bay K
Day 4 Day 6 Day 8
80 s
70 s
MSC
** **
** ** **
**
**
**
**
**
*
*
** **
*
*
CSC MSC CSC MSC CSC MSC
200
400
600
[Ca2]i (nM) [Ca2]i (nM)
800
1000
1200 Rya ExC
Bay K
Bay K Bay K Bay K
70 s
1F/F0
1F/F0
Day 4
Rya Exc
Day 6
Rya Exc
Day 8
Rya Exc
B
FIG. 3. Functional characterization of L-type Cav1 channel
and RyR [Ca2
]i responses. (A) Amplitudes of ryanodine-
(Rya ExC, 1 µM) and Bay-K (10 µM)-induced [Ca2
]i increases
during neuronal differentiation of carcinoma (CSC) and
mesenchymal (MSC) stem cells, respectively. Upper panel:
Representative traces of [Ca2
]i responses of CSC cells after
application of Rya and then with Bay-K. Lower panels indi-
cate values of ∆[Ca2
]i increases obtained in the presence of
Rya represented with white bars and then with Bay-K rep-
resented with gray bars. (B) Neuronal cells from day 8 of
differentiation from CSC and MSC were induced with KCl
(20 mM) or Bay-K (10 µM) and in the presence or absence of
ryanodine (Rya InhC, 10 M) or nifedipine (5 µM). Upper
panels show a typical [Ca2
]i response observed in a neu-
ronal cell derived from CSC from day 8 of differentiation.
Lower panels: Average [Ca2
]i values from control conditions
and in the presence of the respective inhibitors were plot-
ted as ∆[Ca2
]i measurements. White bars represent CSC and
gray bars represent MSC. Plots were typical of ve indepen-
dent expe rim ents . The total number of cel ls a nalyzed in eac h
experiment was 48–59. Data are presented as mean values
SEM; *p < 0.05, **p < 0.001 compared with control data.
SCD-2008_0289.indd 7 3/10/2009 4:55:43 PM
RESENDE ET AL.8
conditions for cells derived from CSC and MSC, respec-
tively (Fig. 4A and Table 2). NEL-positive cells in Bay-K-
treated cultures increased 36% and 23% relative to control
for CSC- and MSC-DN, respectively. Similar effects were
observed when NPC derived from CSC were stained for
nestin (23% 4.1% of the cells cultured with nifedipine,
32% 3.0% of control cells, and 41% 3.4% of those cul-
tured with Bay-K) and from MSC (23% 3.0% of the cells
cultured with nifedipine, 31% 2.8% of control cells, and
38% 4.0% of those cultured with Bay-K) (Fig. 4A and
Table 2). In agreement, nestin and NEL gene and pro-
tein expression were enhanced as revealed by RT-PCR
and Western-blot experiments when NPC of CSC or MSC
had been differentiated in the presence of either DHPs
(Fig. 4B,C, see Table 2 for summary). These results suggest
Role of L-type Ca2 channels in inducing NPC cells
differentiation
In order to investigate the role of Cav1-me diated Ca 2
in ux in promoting neuronal differentiation of NPCs, cells
from the fourth day of differentiation were cultured for 48
h in the presence of an antagonist (5 µ nifedipine) and of
an agonist of L-type channels (10 µ Bay-K). The percentage
of NEL-positive and nestin-positive cells, the percentage of
cells exhibiting [Ca2
]i transients in response to KCl stimu-
lation, and the amplitude of the recorded signals were then
determined. Confocal [Ca2
]i-imaging experiments were
performed after DHP washout.
The percentage of NEL-positive stained cells treated
with nifedipine dropped 62% and 54% relative to control
100
A
B
CE
Nestin NEL
75
50
% of positive cells
25
0
100
75
50
25
0
Conventional
PCR
% of neurons with
Ca2 transients
Nestin
NEL
-actin
Nestin
NEL
-actin
CSC MSC
**
*
*
*
*
** **
**
** **
*
*
**
**
Control Nifedipine
CSC CSCMSC MSC
CSC MSC
Control Nifedipine Bay K
Control Nifedipine Bay K
CSC CSCMSC MSC
D3
2
1
0
F/F0
Bay K
Control Nifedipine Bay K
CSC MSC
CSC MSC
Western Blot
FIG. 4. Cav1-med iate d C a2 in ux induces neuronal progenitor cells (NPCs) to differentiate into neuronal cells. NPCs
from Day 4 of differentiation derived from carcinoma (CSC) and mesenchymal (MSC) stem cells were cultured for 2 days
in the presence or absence of nifedipine (5 µ) or Bay-K (10 µ), and the percentage of nestin-positive (represented as gray
bars) or NEL-positive (represented as dark-gray bars) cells was analyzed by immuno uorescence, counted, and plotted as
a percentage of total cells (A). Gene (B) and protein (C) expression of NEL and nestin from NPC from CSC and MSC at the
same conditions were also evaluated. T he percentage of cel ls exhibiting [Ca 2
]i transients in response to KCl stimulation was
determined (D) and the amplitude of the recorded signals was then evaluated (E).
SCD-2008_0289.indd 8 3/10/2009 4:55:44 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 9
No signi cant effect on cell viability evaluated by Tunel was
observed on treated cells (data not shown), supporting the
idea that a functional neuronal phenotype may be main-
tained via activation of L-type Cav1 channels.
RyR direct neural differentiation process to neuronal
cell fate
To evaluate if the increased neuronal differentiation in
NPC derived from CSC and MSC is directly related to RyR
function, neuronal differentiation was analyzed by adding
1 µM (Rya ExC, which excites RyRs) or 10 µM ryanodine (Rya
InhC, an inhibitor of RyRs). Treatment of cells in the fourth day
of differentiation from CSC and MSC during 48 h with ryano-
dine ExC resulted in a signi cant increase in the proportion
of NEL-positive cells while ryanodine InhC decreased this
percentage to a level signi cantly smaller than that found in
control conditions (see Table 2 for a summary). Likewise, the
percentage of nestin-positive cells increased and decreased
with ryanodine ExC or InhC treatment, respectively (Table 2).
In order to further determine if the reduced number of
NEL-positive cel ls in ryanod ine I nhC-treated c ultu res is due
to the in hibition of neuronal dif ferentiation or/and decreased
cell viability, double-immunelabeling for BrdU and NEL or
BrdU and nestin were performed for both, ryanodine ExC
and InhC concentrations. BrdU would mark the prolifer-
ative cells that are neural progenitor cells (nestin-positive)
or immature neuron cells (NEL-positive). It was found that
ExC treatment promoted an increase on BrdU/NEL-positive
cells for both, CSC- and MSC-DN, compared to control and
that Cav1 channels also participate in induction of differ-
entiation of NPC.
To quantify cell proliferation, NPC on the fourth day
were used in a BrdU assay. No signi cant differences were
observed on BrdU incorporation between nontreated cells
from CSC- and MSC-DN, even when cells were exposed to
nifedipine or Bay-K (Table 2). Based on our analysis of 20–23
randomly selected 20 elds for each experimental condition,
the total number of cells in control, nifedipine-, and Bay-K-
treated cultures were not signi cantly different. These nd-
ings suggest that NPC proliferation in CSC and MSC are
not signi cantly affected by either of the DHPs, suggesting
a common role for the CSC and MSC differentiation and
proliferation.
In Ca2-imaging studies performed on neuronal cells
from the sixth day of differentiation, KCl stimulation caused
very weak increases in uorescence (F/F 0.8 0.23 and
1.0 0.42, for CSC- and MSC-DN, respectively) when these
cells were cultured with nifedipine, whereas those cells cul-
tured in control conditions displayed signals with a mean
amplitude of 1.9 0.29 and 2.2 0.37, for CSC- and MSC-DN,
respectively. These were enhanced in the presence of Bay-K
(2.5 0.31 and 2.6 0.42, i.e., 29% and 20% larger than in
CSC- and MSC-DN controls, respectively) (Fig. 4D). The per-
centage of nifedipine-treated NPC cells responding to KCl
stimulation was also signi cantly reduced 31% 4.8% and
38% 7.5%, respectively. In contrast, in NPC cultured in the
presence of Bay-K, [Ca2
]i transients were observed in 74%
13.4% and 78% 10.7% versus 43% 6.4% and 57%
9.8% of CSC- and MSC-DN controls, respectively (Fig. 4E).
T 2. P P C S NEL, N, BU/NEL, BU/N, R,
N P C (NPC) C (CSC) M (MSC) S C
Drugs
NPC from CSC NPC from MSC
NEL Nestin BrdU/NEL BrdU/nestin NEL Nestin BrdU/NEL BrdU/nestin
Control 61 3.8 32 3.0 13 2.4 19 2.4 69 5.2 31 2.8 12 1.9 23 3.3
Nifedipine 38 4.7** 23 4.1* 12 2.1 19 2.9 37 3.1** 23 3.0* 12 1.5 19 2.4
Bay-K 83 5.7* 41 3.4* 15 2.6 21 2.7 85 4.3* 38 4.0* 14 2.1 22 3.1
Rya InhC 30 3.4** 20 2.3* 6 0.5* 11 1.8* 33 3.2** 21 2.5* 7 0.8* 12 1.7*
Rya ExC 81 6.2* 39 3.6* 22 2.6* 31 3.0* 84 6.7* 40 3.6* 22 1.8* 33 2.8*
AdA 79 6.1* 31 3.5 22 1.9* 32 2.1* 81 6.9* 31 3.5 14 1.9 31 2.0*
XeC 35 2.9** 30 2.5 11 1.2 19 1.7* 32 2.8** 29 2.4 9 1.0* 19 2.1
AdA Rya InhC 59 5.7 19 1.5* 7 0.5* 13 1.8* 69 5.8 22 1.8* 6 0.4* 11 1.7*
AdA Rya ExC 80 6.8* 40 3.8* 21 2.0* 32 2.7* 85 6.4* 39 3.5* 21 2.4* 33 2.9*
XeC Rya ExC 58 7.3 37 4.6* 18 2.3* 30 2.8* 65 7.4 36 4.1 17 2.1* 31 3.9*
BayK Rya InhC 59 4.6 31 2.8 8 0.6* 9 0.4* 71 5.8 30 2.6 6 0.3* 11 0.9*
BayK Rya ExC 80 6.1* 41 3.7* 21 1.8* 33 2.6* 84 6.8* 41 4.2* 23 1.8* 32 2.8*
Nifedipine Rya ExC 60 5.3 30 2.9 20 2.1* 30 3.2* 72 5.7* 29 3.1 21 2.5* 31 3.2*
KCl 81 7. 2* — — — 8 4 6.4* — — —
KCl Rya InhC 58 4.2 — — — 69 5.3 — — —
KCl Rya ExC 79 6.3* — — — 83 6.1* — — —
Nifedipine KCl
Rya ExC
80 5.9* — — — 83 6.2* — — —
Rya InhC, treatment with ryanodine at inhibitory concentration (10 µM); Rya ExC, treatment with ryanodine at excitatory
concentration (1 µM); AdA, Adenophostin-A (2 nM); xestopongin C (XeC, 10 µM); Nifedipine was used at 5 µM, Bay-K at 10 µM, and KCl
at 20 mM; (—) data not evaluated.
Data presented are means SEM of positive-stained NPC for the neuronal enolase (a mature neuronal marker, NEL), nestin (a
marker for NPC) and BrdU/NEL and BrdU/nestin double-labeling. * p < 0.05, ** p < 0.001.
SCD-2008_0289.indd 9 3/10/2009 4:55:44 PM
RESENDE ET AL.10
of Bay-K (1.0% 0.5% and 0.9% 0.3%) or nifedipine (0.8%
0.4% and 0.7% 0.4%) were not signi cantly different
from control conditions (1.1% 0.6% and 0.8% 0.4%).
These results demonstrate that the activation of L-type
Ca2 channel promotes neuronal differentiation through
its functional coupling with RyRs, and that RyRs affect
proliferation of immature neurons and NPCs.
It has been demonstrated that RyRs can be activated by
mobilization of extracellular Ca2 entry through membrane
Ca2 channels in neurons [37], so we evaluated the role of Ca2
in ux in mediating the neuronal differentiation of NPC from
both, CSCs and MSCs, and characterized the functional cou-
pling between RyRs and the regulatory effects of Ca2 in ux
on neuronal differentiation. We studied the impact of activa-
tion o f VGCCs o n neu rogenes is in cult ure s tre ated w ith r yano -
dine ExC or InhC in the presence of depolarizing extracellular
medium by adding KCl (20 mM). The proportion of NEL-
positive cells was increased in conditions with depolarizing
medium but decreased when it was co-treated with ryanodine
InhC or nifedipine (5 µM) (Table 2). The co-treatment with KCl
and nifedipine in the presence of ryanodine ExC or ryanodine
InhC led to a signi cant increase and decrease, respectively,
on the percentage NEL-positive cells. These data indicate that
RyRs play a common role in neurogenesis in different cell
lines, such as CSCs and MSCs, by increasing [Ca2
]i triggered
by activation of L-type Ca2 channels.
Discussion
Spontaneous [Ca2
]i oscillations constitute a hallmark of
developmental networks. They have been observed in sev-
eral neural structures including the retina [38], neocortex
[39], hippocampus [40], and spinal cord [41]. Although this
spontaneous activity plays a key role in the control of neuro-
nal differentiation and synaptogenesis, very little is known
about the cellular and molecular events underlying these
phenomena during neuronal differentiation.
We further studied the role of [Ca2
]i oscillations in differ-
entiation using embryonal CSCs and adult MSCs. Initially
we demonstrated expression of RyRs in early embryonal
carcinoma and mesenchymal adult stem cells for the rst
time. These receptors and the VGCCs are likely respon-
sible for the calcium-induced calcium release (CICR) con-
trolling the spontaneous [Ca2
]i events. The present data
show a transient CICR mechanism in both CSCs and MSCs,
involving ER receptors (IP3Rs and RyRs) and plasma mem-
brane L-type channels. The presence of classic RyRs was
detected by the caffeine-induced [Ca2
]i increases and its
blockage by Rya InhC (10 M ryanodine). Furthermore,
the caffeine-induced [Ca2
]i responses were abolished by
the L-type channel blocker nifedipine. While N- (-CgTx)
and P/Q-type (-MVIIC) blockers presented no effects on
CSC, a small effect was promoted by the N-type blocker on
MSC. It is worth mentioning that nifedipine does not pass
through the plasma membrane, so it cannot interact directly
with RyRs [42]. Thus, our data reveal a functional coupling
between Cav1 L-type channels and RyRs in neurons [37,43],
but not in undifferentiated CSC and MSC.
The involvement of IP3Rs in the mobilization of calcium
from intracellular stores was also evaluated. The results
demonstrate that XeC completely abolished the [Ca2
]i
increase induced by the SERCA pump inhibitor CPA, thus
decreased on InhC treatment. Likewise, the percentage of
BrdU/nestin-positive cells treated with ExC and InhC was
signi cantly increased and decreased relative to control con-
ditions for CSC- and MSC-DN, respectively (Table 2 and Fig.
5A,B). No sign i cant increases on cell apoptosis were noted
for both, ExC or InhC treatment on BrdU/NEL- or BrdU/
nestin-positive cells (data not shown). These data suggest
that RyR-mediated [Ca2
]i elevations induce differentiation
of NPCs, regulates the proliferation of immature neurons,
and increases the proliferation rate of NPCs.
Furthermore the role of IP3R on neuronal differentiation
and proliferation of NPC from CSC and MSC was evaluated
as well as if there was any correlation with RyR activation or
inhibition. The activity of adenophostin-A (2 nM), an IP3R
agonist, in NPCs, the presence or absence of ryanodine ExC
and InhC were tested. Adenophostin-A and ryanodine ExC
promoted an increase of BrdU/NEL-positive cells compared
to control without any drug, but with similar results when
treated with adenophostin-A alone (Fig. 5B and Table 2). A
signi cant decrease in the percentage of BrdU/NEL-positive
cells in ryanodine InhC treatment was observed, and co-
treatment with ryanodine InhC and adenophostin-A did
not alter this result. This suggests that IP3R participates only
on differentiation but not on proliferation. The percentage
of BrdU/nestin-positive cells treated with adenophostin-A
alone presented an increase on NPC of CSC and MSC rela-
tive to control. However, treating NPCs with adenophostin-
A and ryanodine ExC or InhC induced a signi cant increase
and decrease, respectively, in cells responses (Table 2 and
Fig. 5A). These data suggest that adenophostin-A-mediated
[Ca2
]i elevations induce neuronal differentiation of NPC via
a pathway independent of RyR and have effects on the pro-
liferation of immature neurons and NPC.
RyR guided the L-type Cav1 channel induction
neuronal differentiation
RyRs participate in the control of Ca2 release through
channels in the ER and contribute to the dynamics of Ca2
signaling in neurons. However, it is not clear whether RyR-
or L-type Cav1 channels-induced [Ca2]i responses contrib-
ute to Ca2 signaling during early neuronal development
and if neurogenesis requires Ca2 signaling by RyR or
L-type Cav1 channels. We therefore directly analyzed the
functional relationship of RyR and L-type Cav1 channels
by applying Bay-K 10 µM, a L-type Cav1 agonist, alone and
in the presence of ryanodine ExC or InhC (Fig. 5A,B, and
Table 2 for a summary). We found that in NPC from both,
CSC and MSC cells, Bay-K alone signi cantly increased
the percentage of NEL- and nestin-positive cells, an effect
that was inhibited by the co-treatment with ryanodine
InhC. The double-immunelabeling for BrdU/NEL-positive
and BrdU/nestin only increased or decreased when NPC
were co-treated with Bay-K and ryanodine ExC or InhC,
respectively. Similar results were obtained with 10 µM
nifedipine treatment (Fig. 6A and Table 2), suggesting that
tonic activity of L-type Cav1 channels is important for the
neuronal differentiation in NPC but not for its proliferation.
On the other hand, neither Bay-K nor nifedipine had any
effects on the neuronal differentiation in undifferentiated
CSCs and MSCs (Fig. 6B and Table 2). The percentage of
NEL-positive cells in CSC and MSC cultures in the presence
SCD-2008_0289.indd 10 3/10/2009 4:55:44 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 11
D
BrbU/nestin-positive Cells
C
40
** ** **
** **
** **
**
**
30
20
10
0
Control
AdA
Bay K
AdA
Rya InhC
AdA
Rya ExC
BayK
Rya InhC
BayK
Rya ExC
BrbU/nestin-positive Cells
CSC MSC
CSC MSC
30
** ****
***
** **
**
20
10
0
Control
AdA
Bay K
AdA
Rya InhC
AdA
Rya ExC
BayK
Rya InhC
BayK
Rya ExC
BrdU/nestin
Control
Control
A
B
AdA
Bay K
Ryanodine
1M
Ryanodine
10 M
BrdU/NEL
Control
Ryanodine
1M
Ryanodine
10 M
Neural progenitor cells from MSC
FIG. 5. Activation of ryanodine receptors (RyR) promotes neuronal differentiation and proliferation. (A) Representative
co-immunostaining for BrdU and nestin (protein marker for neuronal progenitor cells of neuronal progenitor cells (NPC)
from carcinoma (CSC) and mesenchymal (MSC) stem cells on the fourth day of differentiation. These cell cultures were
pretreated with excitatory ryanodine (Rya ExC, 1 µM) or ryanodine inhibitory (Rya InhC, 10 µM) concentrations for 30 min
prior to proliferation induction with adenophostin-A (AdA, 2 nM) or Bay-K (10 µM) over a 48-h period. (B) Cells from the
fourth day of differentiation were pretreated with the same drugs as in (A) and were double-immunostained for BrdU and
neuronal enolase (NEL, protein marker for mature neurons, green) markers for additional 48 h. Control experiments were
carried out in the absence and presence of each drug alone. Plotted data is from three independent experiments with each
of them being carried out in triplicates. * p < 0.05, ** p < 0.001 compared with control data. (C) Mean values SEM percent-
age of BrdU/nestin-positive cells per eld of vision (800 m2) were determined for experiments in (A). (D) Mean values
SEM percentage of BrdU/NEL-positive cells per eld of vision (800 m2) were determined for experiments in (B). Twenty
to twenty-three elds of vision per cover slip were counted. The shown data are representative of results obtained in three
independent experiments. Scale bars in (A) and (B), 50 µm. Figures shown in A and B are from NPC of MSC.
indicating that functional IP3Rs are present in undifferen-
tiated CSC and MSC and during their neuronal differentia-
tion. T hese data led us to conclude that IP3Rs are responsible
for early [Ca2
]i elevations in those stem cells. In later stages
of the differentiation process, regulation of [Ca2
]i relied on
both IP3- and ryanodine-sensitive stores.
SCD-2008_0289.indd 11 3/10/2009 4:55:44 PM
RESENDE ET AL.12
Real-time PCR analysis revealed that gene expression of
RyRs and IP3Rs was already present in CSC and MSC in a
lower level with prevalence of the isoforms RyR2 and RyR3,
in accordance with a previous study [44]. While RyR1 and
RyR2 function as Ca2-release channels, it has been recently
de mon str ated t hat RyR3 may ne gatively r egu lat e RyR 2 ac tiv-
ity [45]. In undifferentiated CSC and MSC, before induc-
tion of neuronal differentiation, caffeine induced no [Ca2
]
i responses coinciding with the low level of expression of the
RyR isoforms. It has been suggested that functional interac-
tions between the three isoforms in stem cells may not allow
caffeine-induced [Ca2
]i responses, and that RyR2 expression
indicates a mature functional phenotype as shown in vari-
ous brain regions [34].
In vivo experiments studying spontaneous [Ca2
]i oscil-
lations have demonstrated their implication in the regula-
tion of cell death, axon path nding and synaptic pruning
[46–48]. Therefore, the molecular and functional develop-
mental events reported in this study are likely to be physi-
ologically relevant. Moreover, further investigations on the
mechanisms that control [Ca2
]i in neuronal differentiation
of stem cells will increase our understanding of activity-
dependent mechanisms in the developing CNS or in neu-
ral stem cells from the subventricular zone and the dentate
gyrus, which persist in human and may allow the develop-
ment of strategies for preventi ng neuronal death in degener-
ative disorders [49].
Speci c role of RyR on [Ca2]i signaling during
neuronal differentiation of stem cells
The spontaneous [Ca2
]i elevation in neuronal-differen-
tiated stem cells is mediated by diverse pathways such as
intracellular Ca2 mobilization from Ca2 stores by RyRs or
IP3Rs and through extracellular Ca2 in ux which involves
multiple ligand- and voltage-gated channels [50,51]. It has
been demonstrated that [Ca2
]i oscillations could be gen-
erated by RyRs [52,53]. Our results indicate that neuronal
differentiation from CSCs and MSCs could be mediated by
RyRs and that RyRs have a key role in promoting sponta-
neous [Ca2
]i elevation in both cell lines used in this study.
Interestingly neurogenesis in ryanodine InhC-treated cul-
tures was signi cantly reduced, while activation of RyRs
with caffeine or ryanodine ExC promoted neurogenesis.
The percentage of NEL- and nestin-positive cells is signi -
cantly decreased in cultures from ryanodine InhC-treated
cultures suggesting that neurogenesis of NPCs from CSCs
and MSCs is mediated by Ca2 signaling through RyRs. This
led us to conclude that RyRs affect the generation and pro-
liferation of NPCs and immature neurons. Nevertheless,
Ca2 entry through L-type Ca2 channels is more ef cient
than through other types of Ca2 channels in regulating
gene transcription, and thus, in playing a critical role in
coupling excitation and transcription in neurons [54]. We
also demonstrated that Cav1 channel modulators can affect
Ca2 in ux through VGCCs during neuronal differentia-
tion. The number of NEL- or nestin-positive cells increased
after exposure to the L-type channel agonist Bay-K or to the
depolarizing agent KCl, while the percentage of positive
cells decreased after exposure to the L-type channel antag-
onist nifedipine. Furthermore, in the presence of nifed-
ipine, the NEL- and nestin-positive cells either had a very
low [Ca2
]i in ux or had no calcium in ux in response to
Control
Control
Nifedipine
XeC
A
Neural Progenitor
Cell from MSC
BrdU and
NEL
Ryanodine
1M
25 CSC MSC ** **
**
Control
CSC
SSEA-1
-Actin
Bay-K
Nifedipine
MSC CSC
Undifferentiated Cells
B
MSC
Nifedipine Nifedipine
Rya ExC
Xec
Rya ExC
XeC
BrdU/NEL-positive Cells
20
15
10
5
0
FIG. 6. Effects of IP3Rs and L-type Ca2-channels inhibi-
tion on ryanodine receptors activation. (A) Representative
co-immunostaining for BrdU and neuronal enolase (NEL,
protein marker for mature neurons) of neuronal progenitor
cells (NPC) from carcinoma (CSC) and mesenchymal (MSC)
stem cells on the fourth day of differentiation. These cell
cultures were pretreated or not with excitatory ryanodine
(Rya ExC, 1 µM) for 30 min prior to proliferation inhibition
with nifedipine (10 µM) or xestopongin C (XeC, 10 µM) over
a 48-h period. Control experiments were carried out in the
absence and presence of each drug alone. Twenty to twenty-
three elds of vision per cover slip were counted. Plotted
data is from three independent experiments in triplicates. *p
< 0.05, ** p < 0.001 compared with control data. Mean val-
ues SEM percentage of BrdU/NEL-positive cells per eld
of vision (800 m
2) were determined. (B) Embryonic CSC
and undifferentiated MSC were incubated in the absence or
presence of Bay-K (10 M) or nifedipine (5 M) under serum-
free conditions. Western-blot experiments were carried out
with protein extracts of these cells for detection of stage-
speci c embryonic antigen-1 (SSEA-1) as a protein marker
for undifferentiated cells and -actin as a constitutively
expressed internal control. The data shown are representa-
tive of results obtained in three independent experiments.
Scale bars in (A), 50 µm.
SCD-2008_0289.indd 12 3/10/2009 4:55:45 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 13
has postdoctoral fellowship supported by FAPESP. A.A. is
acknowledged for suggestions and English editing of the
manuscript. Authors would like to thank Guilherme Higa
for technical assistance and to L.O. Landeira and L.R. Britto
for material and reagents supply.
Author Disclosure Statement
No competing nancial interests exist.
References
1. Banerjee S and G Hasa n. (2005). The InsP3 receptor: its role in neu-
ronal physiology and neurodegeneration. Bioessays 27:1035–1047.
2. Branchaw JL, SF Hsu and MB Jackson. (1998). Membrane excit-
ability and secretion from peptidergic nerve terminals. Cell Mol
Neurobiol 18:45–63.
3. Munaron L, S Antoniotti, A Fiorio Pla and D Lovisolo. (2004).
Blocking Ca2 entry: a way to control cell proliferation. Curr Med
Chem 11:1533–1543.
4. Carafoli E, L Santella, D Branca and M Brini. (2001). Generation,
control, and processing of cellular calcium signals. Crit Rev
Biochem Mol Biol 36:107–260.
5. Ambudkar IS, BC Bandyopadhyay, X Liu, TP Lockwich, B Paria
and HL Ong. (2006). Functional organization of TRPC-Ca
2
channels and regulation of calcium microdomains. Cell Calcium
40:495–504.
6. Schneggenburger R and E Neher. (2005). Presynaptic calcium
and control of vesicle fusion. Curr Opin Neurobiol 15:266–274.
7. Neher E. (1998). Vesicle pools and Ca2 microdomains: new
tools for understanding their roles in neurotransmitter release.
Neuron 20:389–399.
8. Bito H, K Deisseroth and RW Tsien. (1997). Ca2-dependent
regulation in neuronal gene expression. Curr Opin Neurobiol
7:419– 4 29.
9. Grassi C, M D’Ascenzo, A Torsello, G Martinotti, F Wolf, A
Cittadini and GB Azzena. (2004). Effects of 50 Hz electromag-
netic elds on voltage-gated Ca2 channels and their role in
modulation of neuroendocrine cell proliferation and death. Cell
Calcium 35:307–315.
10. Nicotera P and S Orrenius. (1998). The role of calcium in apopto-
sis. Cell Calcium 23:173–180.
11. Spitzer NC, X Gu and E Olson. (1994). Action potentials, calcium
transients and the control of differentiation of excitable cells.
Curr Opin Neurobiol 4:70–77.
12. Meldolesi J. (2001). Rapidly exchanging Ca2 stores in neurons:
molecular, structural and functional properties. Prog Neurobiol
65:309–338.
13. Patterson RL, D Boehning and SH Snyder. (2004). Inositol
1,4,5-trisphosphate receptors as signal integrators. Ann Rev
Biochem 73:437–465.
14. Verkhratsky A. (2004). Endoplasmic reticulum calcium signal-
ing in nerve cells. Biol Res 37:693–699.
15. Ditlevsen DK, LB Kohler, MV Pedersen, M Risell, K Kolkova,
M Meyer, V Berezin and E Bock. (2003). The role of phosphati-
dylinositol 3-kinase in neural cell adhesion molecule-mediated
neuronal differentiation and survival. J Neurochem 84:546–556.
16. Ille F, S Atanasoski, S Falk, LM Itt ner, D Marki, S Buchmann-
Moller, H Wurdak, U Suter, MM Taketo and L Sommer. (2007).
Wnt/BMP signal integration regulates the balance between
proliferation and differentiation of neuroepithelial cells in the
dorsal spinal cord. Dev Biol 304:394–408.
17. Lange C, E Mix, K Rateitschak and A Rolfs. (2006). Wnt signal
pathways and neural stem cell differentiation. Neurodegener
Dis 3:76–86.
18. Resende RR, AS Alves, LR Britto and H Ulrich. (2008). Role of
acetylcholine receptors in proliferation and differentiation of
P19 embryonal carcinoma cells. Exp Cell Res 314:1429–1443.
membrane depolarization. This let us to hypothesize that
cells may express neuronal markers without acquiring a
mature neuronal phenotype [55,56]. The marked inhibition
of NPC differentiation induced in our model by nifedipine
pretreatment suggests that Ca2 in ux through non-Cav1
channels does not play a signi cant role in neuronal differ-
entiation. The importance of Cav1 channels in neurogenesis
is also re ected by their involvement in the regulation of
nestin. Thus, the data obtained with antagonists indicate the
involvement of L-type channels through ryanodine recep-
tors in the development of functional neuronal cells in CSC
and MSC. Further investigations on the detailed pharmacol-
ogy of these receptors were beyond the scope of the present
study. The contribution of each RyR subtype (RyR1, RyR2,
and RyR3) could be identi ed with in vitro differentiation
experiments in which expression of these receptors is spe-
ci cally downregulated by RNA interference (RNAi). Th is
approach could be used to knock down gene expression of
speci c ryanodine receptors and in different combinations.
Further possibilities in studying the functions of RyRs lie
in the development of DNA or RNA aptamers as ryanodine
receptor subtype-speci c inhibitors [57].
Consistent with these results, we found that although
caffeine and ryanodine or KCl and Bay-K still evoked
low-level [Ca2
]i activity in ryanodine InhC- or nifedipine-
treated cultures, this low-level [Ca2
]i activity is not involved
in the activity-induced neurogenesis in undifferentiated
CSC and MSC. Thus, an antagonist (nifedipine) and an
agonist (Bay-K) of L-type Ca2 channels were effective in
inhibiting and promoting neurogenesis, respectively, only
in control and ryanodine ExC culture conditions, but not
in ryanodine InhC-treated cultures. These results demon-
strate that activity-induced Ca2 in ux through L-type Ca2
channels, if not powered by a CICR mechanism, is not suf -
cient to promote neurogenesis. Moreover, our data show that
RyRs and IP3Rs play, respectively, a major and minor role
in mediating the activity-dependent proliferation induced
by spontaneous- or neurotransmitter-induced [Ca2
]i eleva-
tion, while neurogenesis was mediated by both RyRs and
IP3Rs. It is known that neurotransmitters, through activation
of their respective receptors, trigger speci c patterns of cal-
cium transients inside the cell. This process is a prerequi-
site for the progress of neuronal differentiation [47]. Various
receptor subtypes are activated by the same ligand although
coupled to different second messengers. These receptors
may induce divergent signaling in some cells while activat-
ing convergent signaling in other cells. We have previously
shown that for cholinergic [18,19] and purinergic receptors
[58] the nal pathway that induces neurogenesis or pro-
liferation is that mediated by IP3Rs and RyRs. Thus, path-
ways dependent on different neurotransmitters or growth
factors would mobilize Ca2 from ER stores through IP3Rs
and RyRs. Interestingly, the frequency and amplitude of cal-
cium events can be modulated, directing the acquisition of a
de ned neuronal phenotype.
Acknowledgments
This work was supported by Instituto do Milenio,
CNPq-MCT and Rede Nano, CNPq (Conselho Nacional de
Desenvolvimento Cientí co e Tecnológico), and FAPESP
(Fundação de Amparo à Pesquisa do Estado de São
Paulo), Brazil. R.R.R. and E.L. are CNPq fellows. A.H.K.
SCD-2008_0289.indd 13 3/10/2009 4:55:46 PM
RESENDE ET AL.14
varicosities of NG108–15 (rodent neuroblastoma x glioma
hybrid) cells. J Physiol 529 Pt 2:307–319.
36. Sacchetto R, KD Cliffer, P Podini, A Villa, BN Christensen and
P Volpe. (1995). Intracellular Ca2 stores in chick cerebellum
Purkinje neurons: ontogenetic and functional studies. Am J
Physiol 269:C1219–C1227.
37. Chavis P, L Fagni, JB Lansman and J Bockaert. (1996). Functional
coupling between ryanodine receptors and L-type calcium
channels in neurons. Nature 382:719–722.
38. Feller MB, DA Butts, HL Aaron, DS Rokhsar and CJ Shatz.
(1997). Dynamic processes shape spatiotemporal properties of
retinal waves. Neuron 19:293–306.
39. Garaschuk O, J Linn, J Eilers and A Konnerth. (2000). Large-
scale oscillatory calcium waves in the immature cortex. Nat
Neurosci 3:452–459.
40. Ben-Ari Y, E Cherubini, R Corradetti and JL Gaiarsa. (1989).
Giant synaptic potentials in immature rat CA3 hippocampal
neurones. J Physiol 416:303–325.
41. O’Donovan MJ. (1999). The origin of spontaneous activity in
developing networks of the vertebrate nervous system. Curr
Opin Neurobiol 9:94–104.
42. Gordienko DV and TB Bolton. (2002). Crosstalk between ryano-
dine receptors and IP(3) receptors as a factor shaping sponta-
neous Ca(2)-release events in rabbit portal vein myocytes. J
Physiol 542:743–762.
43. Schwab Y, J Mouton, S Chasserot-Golaz, I Marty, Y Maulet and
E Jover. (2001). Calcium-dependent translocation of synaptotag-
min to the plasma membrane in the dendrites of developing
neurones. Brain Res Mol Brain Res 96:1–13.
44. Sharp AH, FC Nucifora, Jr, O Blondel, CA Sheppard, C Zhang, SH
Snyder, JT Russell, DK Ryugo and CA Ross. (1999). Differential cel-
lular expression of isoforms of inositol 1,4,5-triphosphate recep-
tors in neurons and glia in brain. J Comp Neurol 406:207–220.
45. Jiang D, B Xiao, X Li and SR Chen. (2003). Smooth muscle tissues
express a major dom inant negative splice varia nt of the type 3 Ca2
release channel (ryanodine receptor). J Biol Chem 278:4763–4769.
46. Gu X and NC Spitzer. (1993). Low-threshold Ca2 current and its
role in spontaneous elevations of intracellular Ca2 in develop-
ing Xenopus neurons. J Neurosci 13:4936–4948.
47. Gu X and NC Spitzer. (1995). Distinct aspects of neuronal differ-
entiation encoded by frequency of spontaneous Ca2 transients.
Nature 375:784–787.
48. Spitzer NC, CM Root and LN Borodinsky. (2004). Orchestrating
neuronal differentiation: patterns of Ca2 spikes specify trans-
mitter choice. Trends Neurosci 27:415–421.
49. Hagg T. (2005). Molecular regulation of adult CNS neurogen-
esis: an integrated view. Trends Neurosci 28:589–595.
50. Berridge MJ. (1998). Neuronal calcium signaling. Neuron
21:13–26.
51. Berridge MJ, P Lipp and MD Bootman. (2000). The versatility
and universality of calcium signalling. Nat Rev Mol Cell Biol
1:11–21.
52. Faure AV, D Grunwald, MJ Moutin, M Hilly, JP Mauger, I Marty,
M De Waard, M Villaz and M Albrieux. (2001). Developmental
expression of t he calcium release c hannels du ring early neurogen-
esis of the mouse cerebral cortex. Eur J Neurosci 14:1613–1622.
53. Narita K, T Akita, M Osanai, T Shirasaki, H Kijima and K Kuba.
(1998). A Ca2-induced Ca2 release mechanism involved in
asynchronous exocytosis at frog motor nerve terminals. J Gen
Physiol 112:593– 609.
54. Mermelstein PG, H Bito, K Deisseroth and RW Tsien. (2000).
Critical dependence of cAMP response element-binding pro-
tein phosphorylation on L-type calcium channels supports a
selective response to EPSPs in preference to action potentials.
J Neurosci 20:266–273.
55. Balasubramaniyan V, AH de Haas, R Bakels, A Koper, HW
Boddeke and JC Copray. (2004). Functionally de cient neuronal
19. Resende RR, KN Gomes, A Adhikari, LR Britto and H Ulrich.
(2008). Mechanism of acetylcholine-induced calcium signaling
during neuronal differentiation of P19 embryonal carcinoma
cells in vitro. Cell Calcium 43:107–121.
20. Wobus AM, T Kleppisch, V Maltsev and J Hescheler. (1994).
Cardiomyocyte-like cells differentiated in vitro from embry-
onic carc inoma cells P19 are characterized by f unctional expres -
sion of adrenoceptors and Ca2 channels. In Vitro Cell Dev Biol
30A:425–434.
21. Resende RR, LR Britto and H Ulrich. (2008). Pharmacological
properties of purinergic receptors and their effects on prolifera-
tion and induction of neuronal differentiation of P19 embryonal
carcinoma cells. Int J Dev Neurosci 26(7):763–777.
22. Resende RR, P Majumder, KN Gomes, LR Britto and H Ulrich.
(2007). P19 embryonal carcinoma cells as in vitro model for
studying purinergic receptor expression and modulation of
N-methyl--aspartate-glutamate and acetylcholine receptors
during neuronal differentiation. Neuroscience 146:1169–1181.
23. Yoshimoto M, T Shinohara, T Heike, M Shiota, M Kanatsu-
Shinohara and T Nakahata. (2003). Direct visualization of trans-
planted hematopoietic c ell reconstitution i n intact mouse organ s
indicates the presence of a niche. Exp Hematol 31:733–740.
24. Sanchez-Ramos JR, S Song, SG Kamath, T Zigova, A Willing, F
Cardozo-Pelaez, T Stedeford, M Chopp and PR Sanberg. (2001).
Expression of neural markers in human umbilical cord blood.
Exp Neurol 171:109–115.
25. Kato T, T Heike, K Okawa, M Haruyama, K Shiraishi, M
Yoshimoto, M Nagato, M Shibata, T Kumada, Y Yamanaka, H
Hattori and T Nakahata. (2006). A neurosphere-derived factor,
cystatin C, supports differentiation of ES cells into neural stem
cells. Proc Natl Acad Sci USA 103:6019–6024.
26. Grynkiewicz G, M Poenie and RY Tsien. (1985). A new gener-
ation of Ca2 indicators with greatly improved uorescence
properties. J Biol Chem 260:3440–3450.
27. Gafni J, JA Munsch, TH Lam, MC Catlin, LG Costa, TF Molinski
and IN Pessah. (1997). Xestospongins: potent membrane per-
meable blockers of the inositol 1,4,5-trisphosphate receptor.
Neuron 19:723–733.
28. Dayanithi G, I Mechaly, C Viero, H Aptel, S Alphandery, S
Puech, F Bancel and J Valmier. (2006). Intracellular Ca2 regu-
lation in rat motoneurons during development. Cell Calcium
39:237–246.
29. Mak DO, S McBride and JK Foskett. (2001). ATP-dependent
adenophostin activation of inositol 1,4,5-trisphosphate receptor
channel gating: kinetic implications for the durations of cal-
cium puffs in cells. J Gen Physiol 117:299–314.
30. Takahashi M, K Tanzawa and S Takah ashi. (1994). Adenophosti ns,
newly discovered metabolites of Penicillium brevicompactum, act
as potent agonists of the inositol 1,4,5-trisphosphate receptor. J
Biol Chem 269:369–372.
31. Beck A, RZ Nieden, HP Schneider and JW Deitmer. (2004).
Calcium release from intracellular stores in rodent astrocytes
and neurons in situ. Cell Calcium 35:47–58.
32. Tropel P, N Platet, JC Platel, D Noel, M Albrieux, AL Benabid
and F Berger. (2006). Functional neu ronal differenti ation of bone
marrow-derived mesenchymal stem cells. Stem Cells (Dayton,
Ohio) 24:2868–2876.
33. Dent MA, G Raisman and FA Lai. (1996). Expression of type
1 inositol 1,4,5-trisphosphate receptor during axogenesis and
synaptic contact in the central and peripheral nervous sys-
tem of developing rat. Development (Cambridge, England)
122:1029–1039.
34. Mori F, M Fukaya, H Abe, K Wakabayashi a nd M Watanabe. (2000).
Developmental changes in expression of the three ryanodine
receptor mRNAs in the mouse brain. Neurosci Lett 285:57–60.
35. Ronde P, JJ Dougherty and RA Nichols. (2000). Functional
IP3- and ryanodine-sensitive calcium stores in presynaptic
SCD-2008_0289.indd 14 3/10/2009 4:55:46 PM
INTRACELLULAR Ca2 REGULATION OF STEM CELLS 15
Address reprint requests to:
Prof. Rodrigo R. Resende
Department of Physics, Institute of Exacts Sciences
Federal University of Minas Gerais, Belo Horizonte
Avenue Antônio Carlos, 6627, 31270-901
Belo Horizonte, MG
Brazil
E-mail: resende@icb.usp.br or rrresende@hotmail.com
Received for publication September 27, 2008; accepted
after revision November 25, 2008. Prepublished on Liebert
Instant Online November 26, 2008.
differentiation of mouse embryonic neural stem cells in vitro.
Neurosci Res 49:261–265.
56. Balasubramaniyan V, N Timmer, B Kust, E Boddeke and S
Copray. (2004). Transient expression of Olig1 initiates the differ-
entiation of neural stem cells into oligodendrocyte progenitor
cells. Stem cells (Dayton, Ohio) 22:878–882.
57. Resende RR Torres HAM, Yuahasi KK, Majumder P, Ulrich H.
(2007). Delivery systems of nucleic acids drugs for in vivo use.
Drug Target Insights 2:183–196.
58. Resende RR, LR Britto and H Ulrich. (2008). Pharmacological
properties of purinergic receptors and their effects on prolifera-
tion and induction of neurona l differentiation of P19 embryona l
carcinoma cells. Int J Dev Neurosci 26:763–777.
SCD-2008_0289.indd 15 3/10/2009 4:55:47 PM
SCD-2008_0289.indd 16 3/10/2009 4:55:47 PM
